Research ArticleRelationship between rs854560 PON1 Gene Polymorphism andTobacco Smoking with Coronary Artery Disease

Joanna Iwanicka,1 Tomasz Iwanicki,1 Paweł Niemiec,1 Tomasz Nowak,1 Jolanta Krauze,2

WładysławGrzeszczak,3 Sylwia Górczyńska-Kosiorz,3AnnaOchalska-Tyka,4 and Iwona Żak1

1Department of Biochemistry and Medical Genetics, School of Health Sciences in Katowice, Medical University of Silesia,Medyków Street 18, 40-752 Katowice, Poland21st Department of Cardiac Surgery/2nd Department of Cardiology, American Heart of Poland, S. A. Armii Krajowej Street 101,43-316 Bielsko-Biala, Poland3Department of Internal Medicine, Diabetes and Nephrology, School of Medicine and Division of Dentistry in Zabrze,Medical University of Silesia, 3 Maja Street 13-15, 41-800 Zabrze, Poland4Regional Center of Blood Donation and Blood Treatment in Racibórz, Sienkiewicza Street 3, 47-400 Racibórz, Poland

Correspondence should be addressed to Joanna Iwanicka; jiwanicka@sum.edu.pl

Received 23 June 2017; Revised 25 August 2017; Accepted 14 September 2017; Published 29 September 2017

Academic Editor: Michele Malaguarnera

Copyright © 2017 Joanna Iwanicka et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Paraoxonase-1 (PON1) is the antioxidant marker of high-density lipoproteins protecting against atherosclerosis and coronaryartery disease (CAD) phenotype. The purpose of the present study was to determine whether the PON1 gene rs854560polymorphism (163T>A) is associated with CAD in Polish population. rs854560 was genotyped in 494 subjects: 248 patientswith premature CAD and 246 blood donors as a control. We found that the risk of CAD was significantly higher in TThomozygotes than in A allele carriers (OR= 1.87, p = 0 041). The synergistic effect between the TT genotype and cigarettesmoking was observed (SIM= 9.81; SI = 14.70). The relative increase in risk from interaction between factors was over 37(RERI = 36.13). The PON1 polymorphism did not modulate the risk of CAD in response to exposure to other traditional riskfactors. In conclusion, the rs854560 polymorphism may modulate the risk of CAD in response to cigarette smoking in Polishpopulation. Carriers of TT genotype seem to be particularly at risk of CAD, when exposed to cigarette smoking.

1. Introduction

Coronary artery disease (CAD) is one of the main causes ofdeath worldwide. Oxidative modification of low-densitylipoproteins (LDL) in the vessel wall may play a major rolein development of atherosclerosis, which can progress toCAD. Atherosclerosis is characterized by the inflamma-tion build-up of fatty lesions and scarring of arterial wallswith oxidative stress as a primary contributing factor [1].Atherosclerosis is the main reason of CAD which has amultifactorial background and results from numerousinteractions between genetic and environmental factors.Lipid abnormalities, cigarette smoking, overweight/obesity,

male gender, and older age are the main traditional riskfactors for CAD. High-density lipoproteins (HDL) protectagainst CAD, and the paraoxonase 1 (PON1) enzyme pro-vides their main source of antioxidative activity.

PON1 is a calcium-dependent enzyme, responsible forthe hydrolysis of oxidized phospholipids within LDL. Itsaction prevents the accumulation of oxLDL and protectsagainst the initiation and progression of atherosclerosis[2–5]. PON1 is synthesized in the liver and is secreted tothe bloodstream where it bounds HDL through the A1and J apoproteins [6]. It increases the enzyme stability [7].

Low levels, or the complete absence, of PON1 reduce theantioxidative properties of HDL [8]. Therefore, loss of PON1

HindawiDisease MarkersVolume 2017, Article ID 1540949, 7 pageshttps://doi.org/10.1155/2017/1540949

enzymatic activity can promote the manifestation of athero-sclerosis and CAD phenotypes.

PON1 is encoded by the PON1 gene (7q21.3–22.1) [9],which contains approximately 200 single nucleotide poly-morphisms [10], including the 163T>A polymorphism(rs854560). The rs854560 results in the substitution of methi-onine to leucine at amino acid residue 55 (L55M) of thePON1 polypeptide [11]. Several studies have shown that thisPON1 variant could play an important role in Parkinson’sdisease [12], bipolar I disorder [13], ovarian cancer [14],breast cancer [15], and prostate cancer [16]. Moreover, somebut not all previous studies have linked this polymorphismwith CAD [11, 17–21]. The selection of the 163T>A poly-morphism for the study was due to its functional significance(influence of alleles and genotypes on PON1 activity), andthe fact that its role in predisposition to CAD is not entirelyclear (discrepant results of previous case-control studies).Additionally, we searched for potential interactions betweenPON1 alleles and common traditional risk factors of CADincluding cigarette smoking, hypertension, and plasmalipid abnormalities.

2. Materials and Methods

2.1. Subjects.We enrolled 494 Polish Caucasians in our case-control study. All participants were inhabitants of the UpperSilesia region. The patient group included individuals withangiographically confirmed premature CAD (75 femalesand 173 males), aged 44.58± 5.98 years. The control groupconsisted of 246 blood donors with negative familial historyof CAD, aged 43.58± 6.32 years. CAD subjects were recruitedby the same clinician from the First Department and Clinic ofCardiology at the Upper Silesian Center of Cardiology inKatowice and the First Department of Cardiac Surgery atthe Upper Silesian Center of Cardiology in Katowice. Con-trols were selected from blood donors of the Regional Cen-ters of Blood Donation and Blood Treatment in Katowiceand Racibórz.

Inclusion and exclusion criteria, details of the medicalinterview, diagnosis and evaluation, criteria for CAD,myocardial infarction, and risk factors were as describedpreviously [22].

This study was approved by the Ethics Committee ofthe Medical University of Silesia in Katowice (Poland) andinformed written consent was obtained from all subjects.

2.2. Serum Lipid Measurement. Plasma total cholesterol(TC), HDL cholesterol, and triglyceride (TG) levels wereobtained by enzymatic colorimetric methods (Analco,Warsaw, Poland). LDL cholesterol levels were calculatedusing the Friedewald formula [23].

2.3. DNA Extraction and Genotyping. Genomic DNA wasextracted from peripheral leukocytes using the MasterPuregenomic DNA purification kit (Epicentre Technologies,Madison, WI, USA). PON1 polymorphisms were genotypedusing the TaqMan® Pre-designed SNP Genotyping AssayKit (Applied Biosystems, Foster City, CA, USA). The 20μLreaction mix consisted of the following: 1μL template DNA

(15 ng/μL), 10μL TaqMan Genotyping Master Mix (cat.number 4371355), 1μL probe (TaqMan Pre-designed SNPGenotyping Assay), and 8μL deionized water. The probewas diluted in TE buffer (10mM Tris-HCl (pH 8.0) 0.1mMEDTA) (1 : 1) before the reaction. PCR was performedaccording to the manufacturer’s specifications. Genotypingwas performed using a 7300 Real-Time PCR System (AppliedBiosystems). Genotyping was successful in 92% of partici-pants. Genotyping accuracy was checked by regenotyping15% of the samples, and the reproducibility of the resultswas 100%.

2.4. Statistical Analysis. Data were analysed using Statistica12.0 (STATSOFT, Tulsa, OK, USA) and SAS 9.1 (SASInstitute Inc., NC, USA) software. The Shapiro-Wilk testwas used to check the normality of distribution. Comparisonof quantitative data was performed by Mann–Whitney U test(nonnormal distribution) or the Student t-test (normaldistribution). Allele frequencies were deduced from thegenotype distributions. Hardy-Weinberg equilibrium testing,comparisons of genotypes, and allele frequencies betweencases and control subjects were compared by a χ2 test. Statis-tical significance was accepted at p < 0 05. Odds ratios (ORwith 95% confidence intervals) were computed using univar-iate and multiple logistic regression analyses after adjustmentfor age, gender, and traditional CAD risk factors. When thenumber of individuals in any of the analysed subgroups waszero, risk ratio values (95% CI) were used.

To identify potential biological interactions betweenPON1 genotypes and traditional CAD risk factors, the 4 × 2table approach was used. Subjects without the risk allele/genotype, not exposed to specific traditional risk factors, wereused as a reference group (00 code). This group was com-pared to subgroups of subjects exposed to only traditionalrisk factors, only genetic risk factors, and to both traditionaland genetic risk factors (codes 01, 10, and 11, resp.). Synergymeasures in the multiplicative and additive models were usedto interpret the amount of interaction, according to recom-mendations in the literature [24, 25]. Synergy indexes werecalculated on the basis of OR values from the 4 × 2 tables,using the following formulas:

(i) For SIM (multiplicative synergy index),

SIM = OR11OR01 × OR10

1

The ranges of SIM are from 0 to +∞. SIM< 1 meansnegative interaction (antagonism), SIM=1 means no inter-action, and SIM> 1 means positive interaction (synergy).

(ii) For SI (Rothman’s additive synergy index),

SI = OR11 − 1OR01 − 1 + OR10 − 1 2

The ranges of SI and their interpretation are the same asin the case of SIM.

2 Disease Markers

The RERI (relative excess risk due to interaction)parameter represents the relative risk increase resultingfrom interaction between factors, and the AP (propor-tion attributable to interaction) of the combined effectdue to interaction was also calculated, using the followingformulas [24]:

RERI = OR11 −OR10 −OR01 + 1 3

The ranges of RERI vary from −∞ to +∞. RERI< 0means negative interaction, RERI =0 means no interaction,and RERI> 0 means positive interaction.

AP = RERIOR11

4

The ranges of AP are from −1 to +1. AP< 0 means nega-tive interaction, AP=0 means no interaction, and AP> 0means positive interaction.

Asymmetric confidence intervals (CI) for additive inter-action parameters (SI, RERI, and AP) were determined usingthe model of Zou [26].

3. Results

3.1. Study Group Characteristics. The characteristics of CADpatients and blood donors are shown in Table 1. CADpatients had increased total cholesterol, LDL cholesterol,and triglyceride levels and higher body mass index values.Furthermore, the level of HDL cholesterol was significantlylower in CAD patients (Table 1).

3.2. Analysis of the rs854560 PON1 Gene Polymorphism. Thegenotype and allele frequencies of the rs854560 polymor-phism are shown in Table 2. All genotype frequenciesconformed to Hardy-Weinberg equilibrium. The frequencyof the TT genotype was higher in the CAD group than inthe blood donor group (p = 0 041). The risk of CAD wasalmost two times higher in TT homozygotes than in Aallele carriers. This association was not confirmed in themultivariate model after adjustment for traditional riskfactors of CAD.

3.3. The rs854560 PON1 Gene Polymorphism and ClinicalCAD Phenotypes. There were no statistically significantdifferences between rs854560 polymorphism genotypicvariants and myocardial infarction, severe atherosclerosis

Table 1: Clinical and biochemical characteristics of coronary artery disease patient (CAD) and blood donor (BD) groups.

CharacteristicsCADn = 248

BDn = 246

OR∗

(95% CI)p

Age (years), mean± SD 44.58± 5.98 43.58± 6.32 — NS

Male gender, n (%) 173 (69.76) 175 (71.14) 0.94 (0.64–1.38) NS

TC (mmol/L), mean± SD 5.80± 1.35 5.09± 1.21 — <10−6

HDL (mmol/L), mean± SD 1.12± 0.37 1.45± 0.56 — <10−6

LDL (mmol/L), mean± SD 3.88± 1.18 2.99± 1.22 — <10−6

TG (mmol/L), mean± SD 1.86± 0.97 1.37± 0.72 — <10−6

BMI± SD 27.11± 4.24 26.15± 3.84 — 0.01

Familial history of CAD, n (%) 88 (50.57) — 2.54 (2.25–2.86)∗∗ <10−8

Diabetes mellitus, n (%) 22 (8.98) — 2.09 (1.90–2.29)∗∗ <10−8

Hypertension, n (%) 138 (57.26) — 3.24 (2.77–3.78)∗∗ <10−8

Smoking, n (%) 146 (68.54) 67 (31.46) 3.82 (2.62–5.58) <10−7

NS: not statistically significant; OR: odds ratio; SD: standard deviation; TC: total cholesterol; HDL: high-density lipoprotein; LDL: low-density lipoprotein; TG:triglycerides; BMI: body mass index. ∗Univariate analysis. ∗∗Risk ratio values (95% CI), univariate analysis.

Table 2: PON1 rs854560 polymorphism genotype and allele frequencies in patient (CAD) and blood donor (BD) groups.

Genotype/alleleCAD

n = 232 (%)BD

n = 221 (%) Model of inheritance OR (95% CI) p

AA 96 (41.38) 94 (42.53) Dominant versus AT+TT 0.95 (0.66–1.38) NS

AT 103 (44.40) 109 (49.32) Additive versus AA 0.93 (0.63–1.37) NS

TT 33 (14.22) 18 (8.14)Additive versus AA 1.80 (0.95–3.41) NS

versus AA+AT 1.87 (1.02–3.43) 0.04

AA+AT 199 (85.78) 203 (91.86) Recessive versus TT 0.53 (0.29–0.98) 0.04

A 295 (63.58) 297 (67.20) — 0.85 (0.65–1.12) NS

T 169 (36.42) 145 (32.80) — 1.17 (0.89–1.54) NS

NS: not statistically significant; OR: odds ratio.

3Disease Markers

(presence of multivessel coronary disease or critical occlu-sion >90%) observed during coronary angiography, leftventricular hypertrophy, and diabetes mellitus.

3.4. Interactions between rs854560 PON1 Genotypes andTraditional CAD Risk Factors. Potential interactions betweenthe respective rs854560 genotypes and traditional CADrisk factors (male gender, cigarette smoking, hypertension,diabetes mellitus, overweight/obesity, and plasma lipidabnormalities) were analysed.

The synergistic effect was observed between the TTgenotype and cigarette smoking (Table 3). Cigarette smokerswith the TT genotype had an increased risk of CAD than didsmokers with the A allele (AA+AT) and nonsmokers withthe TT genotype.

When analysing the relationship between cigarettesmoking and the rs854560 phenotype using the multiplica-tive model of interaction, the estimated risk of CAD wasalmost 10 times higher than expected (SIM=9.81, 95% CI:1.11–86.97, p = 0 04). When analysing the same interac-tion using the additive model of interaction, the estimatedrisk of CAD was almost 15 times higher than expected(SI = 14.70, 95% CI: 1.74–123.98). In addition, the relativeincrease in risk from interaction between factors was over37 (RERI= 36.13, 95% CI: 1.76–296.93) and the value ofproportion of the combined effect resulting from interac-tion was 0.91 (0.91, 95% CI: 0.13–0.98) (SI = 14.70, 95%CI: 1.74–123.98).

The PON1 polymorphism did not modulate the risk ofCAD in response to exposure to other traditional risk factorssuch as the following: male gender, hypertension, diabetesmellitus, overweight/obesity, and plasma lipid abnormalities(Table 4). Hypertension and diabetes mellitus were excludedfrom the analysis because of lack of these phenotypes inthe control group (factors that prevent being a blood donorin Poland).

3.5. Analysis of the rs854560 PON1 Gene PolymorphismDepending on Smoking Status. To evaluate whether thePON1 polymorphism may be a determinant of smokinghabit, we divided our entire group into smokers and non-smokers. The frequencies of genotypes and alleles are shownin Table 5. There were no differences in genotypic variantdistribution between smokers and nonsmokers (Table 5).

4. Discussion

The multifactorial nature of CAD results from interac-tions between genetic and environmental factors. ThePON1 rs854560 polymorphism potentially modifies therisk of CAD. Here, we show that rs854560 polymorphismTT homozygosity predisposes individuals to CAD in thePolish population.

Association between the TT rs854560 genotype and CADmay be explained by the effects of polymorphism variants onthe activity of paraoxonase 1. The presence of leucine atamino acid residue 55 of the PON1 polypeptide (encodedby the PON1 A allele) is associated with increased enzymaticactivity compared to the 55M phenotype (encoded by thePON1 T allele) [27]. Furthermore, the lowest PON1 activitywas observed in 55MM, TT homozygotes [28]. In the longterm, decreased PON1 enzyme activity can result in thedevelopment and progression of atherosclerosis and CAD[29, 30] due to increased reactive oxygen species andenhanced LDL oxidation [5, 31]. The role of PON1 in inhi-biting of LDL oxidation is very important, and PON1 reducesoxLDL formation by 42–65% [32].

The rs854560 polymorphism has been analysed in popu-lations of different ethnic origins. Our results are consistentwith those previously reported. However, ethnic heterogene-ity and different inclusion/exclusion criteria have led toinconsistent results and conclusions between studies. The Tallele carrier state and TT homozygosity have previouslybeen associated with CAD only in the Turkish population[20, 33]. In most other studies, including those examiningNorth Indian [17], Brazilian Caucasian [34], and otherTurkish populations [35, 36], the AA genotype was identi-fied as a CAD risk factor. Furthermore, the results of otherstudies suggest a protective role of the T allele [18, 19] ora complete lack of association between the rs854560 poly-morphism and CAD [11, 21, 37]. Inconsistencies betweenstudies may be caused by different inclusion criteria ofpatients (e.g., stenosis≥ 70% as a main criterion for inclu-sion [37]) or controls (e.g., the presence of hypertension,diabetes mellitus, and familial history of CAD in controlsubjects [18]).

Here, we also analysed the potential effect of thers854560 polymorphism on the risk of CAD taking intoaccount traditional risk factors. Our results indicate thepresence of a strong synergistic effect between the TTgenotype and cigarette smoking. Cigarette smoke containsa number of oxidizing compounds and is an importantsource of free radicals [38] which contribute to both thedevelopment of atherosclerosis and an increase in the inci-dence of cardiovascular events among smokers [39]. How-ever, it must be noted that a limitation of the presentstudy is the relatively small sample size and no reporteddose of cigarettes. The results presented here should beconfirmed in a larger group of patients, as well as in addi-tional ethnic groups, and should include an additionalinformation about nicotine exposure dose. Daily smokingdose may not only impact on cardiovascular disease butmay also be related to the addictive behaviour [40]. It ispossible that paraoxonase locus may be a determinant of

Table 3: Synergistic effect between the PON1 rs854560 TT genotypeand cigarette smoking (4 × 2 table).

TThomozygosity

Cigarettesmoking

CAD(n = 228)

BD(n = 220)

OR(95% CI)

1 1 22 139.77

(5.26–300.70)

1 0 11 171.17

0.52–2.62)

0 1 117 613.46

(2.29–5.25)

0 0 78 141 1

OR: odds ratio; CAD: coronary artery disease (patient group); BD: blooddonor (control group).

4 Disease Markers

smoking habit through metabolism of xenobiotics presentin smoke [41].

Cigarette smoking is a potent inhibitor of PON1 enzy-matic activity, making the effectiveness of PON1 in smokersparticularly important [42, 43]. It was shown that cigarettesmoke extract decreases PON1 activity by modifying the freethiol groups of the enzyme. It can be explained by the effectsof several of the hundreds of chemical components oftobacco smoke including various reactive aldehydes as wellas aromatic hydrocarbons [43]. Moreover, it is importantthat smoking may increase neutrophil myeloperoxidaseactivity [44], which has been shown to interact with HDLand to modulate paraoxonase 1 function [45]. The numberof neutrophils was found to be higher in current or ex-smokers than in nonsmokers [46]. James et al. [47] foundthat the concentration and activity of PON1 in plasma weresignificantly lower in current smokers than in never smokers.In specific groups of US veterans [48], patients diagnosedwith CAD [47] and inhabitants of the Spanish province ofGerona [42], a similar downward trend was observed. Fur-thermore, Mouhamed et al. [49] showed that, in smokers,the TT and AA genotypes presented the lowest and highestlevels of PON1 activity, respectively. However, we did notobserve differences in genotype distribution and allele fre-quencies between smokers and nonsmokers. These resultsare not consistent with previously described [49]. As in thecase of association studies, these differences may also bedue to differences in demographics, ethnic origin, matchingcriteria, and coexistence of other addictions. It is also possiblethat the observed differences result from chronic exposure topollutants in Silesian Voivodeship, an industrial area wherethe study groups of the present work were recruited.

5. Conclusions

In conclusion, our results suggest that the rs854560 TTgenotype may be a potent risk factor for CAD in the Polishpopulation. Furthermore, TT homozygotes are particularlysusceptible to the effects of smoking addiction, whichdecreases the activity of this protective and antiatherogenicenzyme. Functional studies support the idea that the TTgenotype promotes the development of atherosclerosis andinfluences the manifestation of CAD, despite the conflictingresults of case-control studies.

Conflicts of Interest

The authors declare no conflict of interest.

Acknowledgments

This research was supported by grants from theMedical Uni-versity of Silesia KNW-2-002/D/7/N and KNW-1-009/K/7/0.

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Table 4: Synergy index (with 95% CI) for interactions between PON1 rs854560 TT genotype and traditional risk factors for CAD.

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Table 5: PON1 rs854560 polymorphism genotype and allele frequencies in smoker and nonsmoker groups.

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